High-Resolution 3D Fluorescent Imaging of Intact Tissues

Abstract

Histological analysis of fluorescently labeled tissues has been a critical tool to understand molecular organization in situ. However, assessing molecular structures within large cells and in the context of human organ anatomy has been challenging because it requires penetration of staining reagents and light deep into opaque tissues, while also conforming to the spatial constraints of high-resolution objective lenses. This methodology article describes optimized sample preparation for sub-micron resolution 3D imaging in human and rodent tissues, yielding imaging depth (>100 µm) and resolution (<0.012 µm 3 voxel size) that has previously been limited to whole-mount in vitro organoid systems, embryos, and small model organisms. Confocal images of adult human and rodent organs, including heart, kidney, and liver, were generated for several chemical and antibody stains in cleared tissue sections >100 µm thick. This method can be readily adopted by any lab performing routine histology and takes 3 days from the start of tissue preparation to 3D images. Contributions D.E. designed the study, performed experiments, analyzed results, and wrote the manuscript. A.M.M. performed cardiac cell therapy surgeries and animal care. X.Y. generated PSC-derived cardiac myocytes for cell therapy. W.R.M., C.E.M., and X.Y. edited the manuscript. W.R.M. and C.E.M. provided supervision and acquired funding. Competing Interests W.R.M. and C.E.M. are scientific founders and equity holders in Cytocardia. The other authors declare no competing interests.

Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry

“…Optical clearing was performed as previously described with some modifications ( 28 , 113 , 114 ). In vivo transcardiac perfusion with 10 to 15 ml of PBS infused over 5 min was used to wash out blood.…”